This proposal aims at using Drosophila to understand the mechanisms that regulate stem-cell-mediated intestinal tissue homeostasis. Billions of cells in the human gastrointestinal (GI) tract are shed and replaced every day. The mechanisms that maintain this homeostasis must be balanced well, and mis-regulation will lead to various diseases including tissue damage, inflammation and cancer. The size and complexity of the human GI tract renders the studying of homeostasis involving multiple cell types and regulatory pathways rather difficult. Similar to the mammalian intestine, the Drosophila midgut represents an active homeostatic system maintained by resident stem cells. Evolutionarily conserved pathways including EGF, Insulin, Wnt, and JAK-STAT regulate midgut intestinal stem cells (ISCs) for tissue growth. We will investigate how the two main epithelial cell types in the midgut, mature enterocytes and differentiating enteroblasts, produce growth factors to modulate ISC division and maintain tissue wellness. The specific aim 1 is to investigate how Rab11 regulates Upd3 expression in mature enterocytes to modulate ISC division. Rab11 is a small GTPase known to control protein trafficking in Golgi and endosomes. Loss of Rab11 function in enterocytes causes a prominent ISC proliferation phenotype, concomitant with an increased expression of the JAK-STAT ligand Upd3. Moreover, this phenotype is very similar to that observed in mouse intestinal epithelium after Rab11a knockout. Genetic and molecular approaches will be used to identify the pathway that connects Rab11 with Upd3. The specific aim 2 is to examine the function of enteroblasts as part of the epithelial niche to regulate ISCs. From an enteroblast-specific pilot RNAi screen we have uncovered the kinase Misshapen interacting with Warts-Yorkie to regulate Upd3 expression. This is conserved in mammalian cells as the MAP4K4-LATS-YAP pathway. We will analyze in detail this pathway and the function of other genes from the genetic screen to establish that enteroblasts also act as an important niche cell type. Our long-term goal is to understand the coordination among epithelial cells to produce the optimal amount of growth factors for intestinal homeostasis. The results obtained should provide important insights into therapeutic strategies for human inflammatory diseases, aging and cancer.